The long range objectives of this project are to understand the mechanism by which ATP synthase complexes (F0F1 ATPases) of higher animals synthesize ATP, and the mechanism by which this process is regulated in both normal and neoplastic tissues. Studies supported by this grant, now in its 21st year, have helped play fundamentally important roles in the elucidation of the complex substructure of F1 (i.e., alpha3 beta3 gamma delta epsilon), recognition and verification of its subunit asymmetry, resolution of its 3-D structure to 3.8A, identification of subunit locations of interacting ligands, and characterization of its interaction with an ATPase regulatory peptide. Future studies will focus on novel approaches designed to elucidate the relationship of the complex structure of F1 to both its catalytic and regulatory mechanisms. Specific aims are 6-fold: 1) Assess the functional and regulatory significance of beta subunit asymmetry within the F1 moiety of the ATP synthase complex. 2) Characterize the adenylate kinase homology regions predicted to form an ATP binding "pocket" within F1 beta subunits by evaluating directly their role in nucleotide binding. 3) Establish whether critical amino acids in F1-beta subunits labelled with the heavy atom iodine lie within the adenylate kinase homology regions or within the "essential amino acid rich" C-terminal region. Assess their importance for F1-beta function. 4) Determine the external accessibility of both the adenylate kinase homology regions and the "essential amino acid rich" C-terminal region of F1-beta subunits before and during catalysis. 5) Characterize the interface between F0 and F1 in greater detail with the objective of identifying the subunit site of action of the novel probe diethylstilbestrol. 6) Evaluate the roles of two newly discovered "regulatory" peptides of F0F1 in normal and neoplastic tissues, and identify their site(s) of action. To accomplish these goals, a combination of biochemical; molecular biological; and immunochemical technologies, all established procedures in the P.I.'s laboratory, will be employed. These ongoing studies are both necessary and fundamental to our eventual understanding of the mechanism and regulation of mitochondrial ATP synthase complexes in normal and neoplastic tissues. Five years of support is requested.